1. Field of the Invention
The present invention provides recombinant insulin-like growth factor (IGF) expression systems that yield high levels of product which are easily processed to provide IGF with a proper N-terminus for human use.
2. Description of the Related Art
Recombinant production allows the large scale manufacturing of therapeutic proteins while avoiding many of the difficulties and hazards of protein purification from natural sources. In the case of human proteins, recombinant manufacturing is frequently the only practical method for producing the amounts of protein required for commercial sales of therapeutic products. Recombinant production also eliminates worker exposure to human fluid and tissues, avoiding potential exposure to infectious agents such as viruses.
Recombinant manufacturing involves the expression of a DNA construct encoding for the desired protein in a recombinant host cell. The host cell can be either prokaryotic (e.g., bacteria such as Escherichia coli) or eukaryotic (e.g., yeast or mammalian cell-line). For large scale recombinant manufacturing, bacterial or yeast host cells are most commonly used, due to the ease of manipulation and growth of these organisms and also because these organisms require relatively simple growth media.
Recombinant manufacturing, however, does have its difficulties. Expression constructs must be optimized for a particular protein and for a particular host cell. Expressing a recombinant protein in a host cell exposes the recombinant protein to a new set of host cell enzymes, such as proteases, which can modify or even degrade the recombinant protein. Modification and degradation of the recombinant protein is undesirable, as it decreases yields and can complicate the purification of the recombinant protein. Polypeptides over-expressed in the bacterial cytoplasm often accumulate as insoluble “inclusion bodies” (Williams et al., Science 215:687-688 (1982); Schoner et al., Biotechnology 3:151-154 (1985)). Polypeptides accumulated in the form of inclusion bodies are relatively useless. Conversion of this insoluble material into active, soluble polypeptide requires slow and difficult solubilization and refolding protocols that often greatly reduce the net yield of biologically active polypeptide. This problem has particularly impacted the production of IGF, resulting in numerous attempts to solve the “refolding problem.” Even when polypeptides are expressed in the cytoplasm of bacteria in soluble form, they often accumulate poorly as a result of degradation by host proteases. Furthermore, the accumulated polypeptides often lack the desired amino terminus. This problem is commonly addressed by the expression of a fusion protein, in which the N-terminus of the desired polypeptide is fused to a carrier protein.
However, the use of fusion polypeptides has drawbacks. It is often necessary to cleave the desired polypeptide away from the fusion partner by enzymatic or chemical means. This can be accomplished by placing an appropriate target sequence for cleavage between the fusion partner and the desired polypeptide. Unfortunately, the enzymes most widely used for polypeptide cleavage are expensive, inefficient, or imprecise in their cleavage and cannot be successfully applied to a majority of fusion constructs. For example, enterokinase and Factor Xa are mammalian enzymes that are expensive to produce and exhibit highly variable cleavage efficiency. Meanwhile, enzymes like subtilisin are relatively inexpensive to produce, but their precision is unacceptable for commercial-scale processes under current “Good Manufacturing Practices” (GMP). The human rhinovirus 14 protease, termed 3C protease, is a robust, precise, and inexpensive enzyme that cleaves the amino acid sequence E-(V or T)-L-F-Q-G-P (SEQ ID NO: 22) immediately N-terminal to the glycine residue. 3C protease has been used to cleave IGF from dsbA-IGF fusion constructs (Olson et al., Protein Expr Purif. 14:160-166 (1998)). As a substantial portion of the dsbA-IGF is refractory to cleavage, however, 3C protease may not be universally applicable to 3C site containing IGF fusion constructs.
The patent literature describes numerous fusion systems for the production of IGF, each of which has certain advantages and disadvantages. For example, constructs encoding native mature IGF-1 (with an additional methionine) yield low expression levels. A variety of approaches were developed to enhance expression, including the use of fusion partners (Schulz M. F. et al., J. Bact. 169:585-53921(1987)), and the use of multiple protease-deficient hosts (Buell et al., Nucleic Acids Res. 13:1923-1938 (1985)). However, the growth characteristics of these protease deficient hosts are not ideal for high intensity fermentation. It also has been found that good expression can be obtained simply by adding Met-Arg-Lys to the N-terminus, but this does not produce authentic IGF-1 (Belagaje et al. Protein Sci. 6:1953-19623 (1997)).
An additional problem that is often overlooked when designing IGF fusion constructs is that the fusion partner is essentially a waste product. Prokaryotes can express only a finite amount of recombinant protein—typically up to 40% of the dry mass of the cell. If the fusion partner is a large protein relative to the IGF, then the effective amount of IGF is reduced. For example, dsbA-IGF fusion proteins are approximately 31,500 daltons, of which the IGF is approximately 7,500 daltons, or approximately 24 percent of the total fusion protein. See U.S. Pat. No. 5,629,172. Thus, the efficiency of IGF protein production can be enhanced by using a proportionally smaller fusion partner.
Identifying an effective fusion partner is a difficult task that typically involves significant trial and error. Therefore, improved constructs with more effective fusion partners are required to enhance the production of recombinant IGF.